Mon, Sep 27 21

Gyrase activity assay with chloroquine gels #

Revised Gyrase activity assay #

Revised the Gyrase activity assay to include heat killing each enzyme after 37C incubation periods.

Reaction setup #

Setup this reaction starting with pFC9 control which included

  • 3.33 pFC9 (1ug)
  • 6 ul Gyrase buffer (5x)
  • 3 ul rCutsmart (10x)
  • 3 ul 10x ATP for EcoP1rI (NEB product code B61015)
  • 14.67 ul H20

20 ul of this formed the Topo treated fraction to which I added 1ul Topo. After incubation and heatkill fractioned 10ul and added 1 ul DNA Gyrase and incubated + heatkilled. I set up two replicates of these reactions so I could run two independent chloroquine gels.

Chloroquine gel #

Fred suggested running reaction products on chloroquine gel to better resolve supercoiling. Gel protocol is below

  1. Prepare a 1% agarose gel with 2x TBE buffer
  2. Allow gel to cool to around 50C.
  3. Add chloroquine to a final concentration of 2.5 ug / ml of agarose
  4. Pour gel.

Made two 12 cm 100 ml 1% agarose with .250g chloroquine each using 2x TBE buffer. Running gels for 16 hours at 110V according to supplemental materials in Robert’s paper.